5.3.1. The rearing method

The
rearing method used for this test is detailed in Crailsheim et al. (2013), summarized in Fig. 9, and
outlined in the steps below.

1) For one replicate, larvae are collected
preferably from a unique colony. If two colonies are necessary, larvae
originated from both colonies must be distributed in two samples of equal size
(24 larvae) in each plate. The colonies have to be healthy and must not show
any visible clinical symptoms of pests, pathogens (see BEEBOOK Volume II)
and/or toxin stress.

2) Tests are performed with summer larvae during a
period from the middle spring to the middle autumn (the exact time of year
varies by location).

3) In case of sanitary treatment (i.e. products
added to the hive for purposes of disease/pest control), the date of
application and the kind of product has to be noted. No treatment should be
applied within the 4 weeks preceding the beginning of experiments.

4) The queen is confined in its own colony in an
excluder cage containing a comb with emerging worker brood and empty cells for
less than 30 hours in order to obtain a large number of fresh laid eggs.
According to queen vigour, the queen’s isolation time can be reduced in order
to minimize variability in larval size (age).

5) To ensure one obtains enough larvae, it is
recommended to isolate the queens in 2 or 3 colonies in the eventuality that
one queen lays few or no eggs.

6) The queen is removed from the cage and the caged
comb is left in the hive for 3 days until the larvae hatch.

7) At day 1 (D1, Fig. 9), the comb containing fresh
laid eggs is carried from the hive to the laboratory (regulated at a constant
temperature of 25°C if possible), in a special wooden container in order to
avoid temperature variation and to transfer the larvae into individual rearing
cells. We recommend crystal polystyrene grafting cells (ref CNE/3, NICOPLAST
Society), having an internal diameter of 9 mm.

8) Before use, the cells are submerged for 30 min in
0.4% MBC (methyl benzethonium chloride) in water, and then dried in a laminar-flow
hood. MBC can be replaced by chloride tablets generally used for nursing bottle
sterilisation.

9) Each cell is placed into a well of a 48-well
tissue culture plate, which was previously half filled with a piece of dental
roll wetted with 15.5% glycerol in 0.4% MBC.

10) The young larvae are transferred with a grafting
tool (a thin paint brush for example) from the frame into individual plastic
cells previously filled with 20 μl of diet A (Table 8).

11) The larvae are fed once a day (except day 2)
with a micropipette. Diet composition varies according to larval age (Fig. 9, Table
8). The diet is warmed at 34°C prior to each use.

12) The plates are placed into a hermetic Plexiglas
desiccator (NALGENE 5314-0120 or 5317-0180 or similar, according to the required
volume), provided with a dish filled with K2SO4 saturated solution in order to
maintain a water-saturated atmosphere.

13) The desiccator is placed into an incubator at 34
± 0.5°C. This parameter is crucial considering that susceptibility to a compound
may vary significantly according to temperature (Medrzycki et al.,
2010).

14) At D7 (pre pupa stage), the plates are
transferred into a hermetic container containing a dish filled with a saturated
NaCl solution in order to maintain 80% relative humidity. The container is then
placed into an incubator at 34°C.

15) At D15, each plate is transferred into a crystal
polypropylene box (11 x 15 x 12cm) with a cover aerated with a wire mesh, and
containing a piece of comb with a small plastic royal pheromone diffuser in its
centre (Bee Boost®), fixed with a wire.

16) Emerging bees are fed with syrup and pollen
powder delivered using bird feeders or similar structures. The boxes are kept
in the hermetic container.